Azure 600

The whole cell lysates were prepared by using RIPA Lysis and Extraction Buffer (Thermo Scientific, IL, USA) with Pierce Protease and Phosphatase Inhibitor Mini (Thermo Scientific, IL, USA). The fractionated lysates were prepared by using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, IL, USA). Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad; CA, USA), and the concentration in individual samples was equalized before adding 4x Laemmli buffer to a final concentration of 1x. Equal amounts of protein (50 μg whole cell lysates, 45 μg fractionated lysates) were run on 10% SDS-PAGE gels and then transferred with iBlot 2 Transfer Stacks and iBlot 2 Dry Blotting System onto PVDF membranes (Invitrogen, Thermo Scientific, IL, USA). After one hour 5% milk block, membranes were probed with the antibodies diluted with 5% BSA (NRF3) or 5% milk (β-actin and PCNA and secondary antibodies) in Tris-buffered saline with 0.1% Tween 20. Primary antibodies anti-NRF3 (HPA055889, Sigma-Aldrich), anti-β-actin (NB600-501, Novus Biologicals, UK), and anti-PCNA (NB600-501SS, Novus Biologicals, UK) were incubated overnight, and appropriate HRP-conjugated secondary antibodies (sc-2054 and sc-2055, Santa Cruz, CA, USA) were incubated at RT for one hour. Blots were detected with the Western blot imaging system Azure 600 (Azure Biosystems, CA, USA).

The experimental procedure was based on the protocol by Sallés and Strickland (1999) [46 (link)], with the following modifications. To keep the entire length of the polyA tail of each transcript, the total RNA was extracted by the phenol-chloroform method. Isolated RNA was incubated with 1 µL of 50 μM oligo-dT per sample for 5 min at 65 °C. The ligation mix was prepared from the following components: T4 ligase (1 µL), Superscript IV 5× buffer (5 µL), 20U/µL RNAse inhibitor (1 µL), 10 mM dNTP (1 µL), 10mM ATP (1 µL), 1M MgCl2 (0.1 µL), 0,1M DTT (2 µL), and RNAse-free water (2 µL). Each sample was processed in 10 µL of the mixture and incubated for 30 min at 42 °C to allow T4-mediated oligo-dT ligation. Anchoring was carried out by incubating with 1 µL of oligo-dT anchor (5′-GCGAGCTCCGCGGCCGCGT-3′) for 1 h at 12 °C, followed by 2 min of incubation at 42 °C. cDNA synthesis was performed by the addition of 1 µL of Superscript II Reverse Transcriptase with the following setup: 45 min at 45 °C, 10 min at 80 °C, hold at 4 °C. Prepared cDNA was subjected to PCR with gene-specific forward primers and anchoring reverse primer (Supplementary Table S1). Images were developed in an Azure 600 (Azure Biosystems). Each experiment was carried out in technical triplicates.

Protein was extracted from the RPE/choroid, according to a previously described methodology [28 (link)] and protein concentrations were measured by Pierce™ BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 10 μg of protein was loaded into each well, separated on a 4–12% Bis-Tris SDS-PAGE gel (NuPAGE, Invitrogen), and transferred to a polyvinylidene fluoride membrane (Bio-Rad). After blocking with 5% bovine serum albumin (BSA, Sigma) in Tris-buffered saline (TBS, Bio-Rad), membranes were incubated overnight with primary antibodies: anti-mouse LCN2/NGAL (1:1000; catalog# AF1857, R&D Systems), or anti-α-Tubulin (1:1000; catalog# T6199, Millipore Sigma), used as a loading control. After three washes with 0.1% Tween-20 in TBS, incubated for 1 h with appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-goat IgG (1:7500; Jackson ImmunoResearch), or HRP-conjugated anti-mouse IgG (1:8000; GE healthcare Lifesciences). Membranes were developed using SuperSignal^™ West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). Images were acquired using an Azure c500 or Azure 600 gel imager with cSeries Capture Software (Azure Biosystems).

Total proteins were extracted by RIPA buffer (Beyotime, China) containing 1nmol/L PMSF (Beyotime). The proteins were resolved by SDS-PAGE and then transferred on a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, USA). After incubation with 5% skimmed milk at room temperature for 2 h, the blot was hybridized with the primary antibody at 4 °C overnight. After washing by TBST three times, the blot was incubated with the secondary antibody (Beyotime, A0208, A0216, dilution: 1:1000) at room temperature for 1 h. After adding the enhanced chemiluminescence (NCM Biotech, China), the immunoblots were obtained using Azure600 (azure biosystems, USA). The primary antibodies used in this study were GAPDH (Abcam, UK, ab181602, dilution: 1:5000), TDP-43 (Abcam, ab190963, dilution: 1:2000), ABHD2 (absin, China, abs140798, dilution: 1:1000), Cleaved-caspase3 (Cell Signaling, USA, #9664, dilution: 1:2000), p53 (Proteintech, USA, 10442-1-AP, dilution: 1:5000), Bax (Cell Signaling, #5023, dilution: 1:1000), Bcl-2(Abcam, ab182858, dilution: 1:2000), Ki67 (Abcam, ab16667, dilution: 1:1000).